The long term goals of this research include the development of methodology to permit the folding, flexibility and dynamics of protein structure to be interrogated, and the development and use of fluorescent probes in novel ways to enhance our understanding of protein structure, dynamics and function. During the five years of proposed research, the focus of efforts will be on study of the mechanism of modified DHFRs through steady state, pre-steady state and single molecule kinetic studies. Fluorescence spectroscopy will be used to study the protein conformational changes and their association with the key steps in DHFR catalysis. PUBLIC HEALTH RELEVANCE: The present project is aimed at describing and quantifying the particular sequence, structural and dynamic properties that operate to enhance enzymatic catalysis. The proposed studies will provide important insights into enzyme function and will foster protein design and drug discovery efforts. The enzyme DHFR, which is the target of important antineoplastic and antimicrobial drugs, will be the focus of our efforts.